Plasmodium falciparum (Pf) malaria remains a major public health threat. A key tool for the control, elimination, and possible eradication of malaria is an effective vaccine. The development of a highly effective malaria vaccine has been hindered in part by a poor understanding of the interaction between Pf and the human immune system. The overall objective of this project, which represents a collaborative effort between scientists at LIG/NIAID and the Malaria Research and Training Center (MRTC) in Bamako, Mali, is to aid malaria vaccine development by generating knowledge of the immune mechanisms and their Pf targets that ultimately provide protection from malaria. To this end we are translating recent advances in basic and applied immunology as well as genomics-based technology to rigorously conducted longitudinal cohort studies in malaria-endemic areas of Mali. In 2011, we conducted studies of the B and T cell and innate immune response to malaria and initiated a new cohort study in which systems immunology tools are being applied to identify signatures of immune responses that protect against malaria. This year we also laid the groundwork for an apheresis protocol in Mali which will allow for more sophisticated phenotypic and functional analysis of the human immune response to malaria. Optimism that a highly effective malaria vaccine can be developed comes in part from the observation that malaria immunity can be acquired through natural infection;however, unlike the immunity that is efficiently acquired after exposure to many pathogens or after vaccination, protection from malaria is only acquired after repeated infections. The immune mechanisms that ultimately confer protection from malaria and the basis for their relatively inefficient acquisition remain unclear. To address these questions we focus on longitudinal cohort studies in Mali where there is a predictable, sharply-demarcated, and intense six-month malaria season followed by a six-month dry season when little to no Pf transmission occurs. This research model of seasonal malaria allows us to design prospective studies in which blood samples are collected at strategic time points before, during and after Pf infection, which in turn allows us to address two general questions: 1) which immune parameters correlate prospectively with protection from malaria, and 2) how does Pf infection modulate the human immune response. Our recent work on the B cell response to malaria illustrates the utility of this model. Previous studies demonstrated that purified IgG from malaria-immune adults, when transferred to children acutely ill with malaria, reduced fever and parasitemia, thus indicating that Abs play a critical role in controlling malaria. However, the antigen specificity of Abs that protect against malaria and the B cell biology that underlies their slow acquisition and rapid loss is poorly understood. To address these questions we analyzed plasma and peripheral blood mononuclear cells (PBMCs) collected longitudinally from children and adults in Mali. For example, we probed plasma collected before and after the six-month malaria season against a protein microarray containing approximately 23% of the Pf 5,400-protein proteome. We found that Ab reactivity to hundreds of Pf proteins rose dramatically in children during the malaria season;however, most of this response appeared to be short-lived based on cross-sectional analysis before the malaria season, which revealed only modest incremental increases in Ab reactivity with increasing age. Ab reactivities to 49 Pf proteins measured before the malaria season were significantly higher in immune versus susceptible children. We are validating these findings in a larger cohort study in Mali (described below) which we initiated in May 2011, and also with collaborators in malaria-endemic areas of Kenya and Angola. This approach may prove to be a useful strategy for understanding fundamental properties of the humoral immune response to malaria and for identifying novel vaccine targets. We also analyzed the memory B cell (MBC) response at various time points relative to the malaria season and dry season and found that Pf-specific MBCs are only acquired gradually in a stepwise fashion over years of repeated Pf exposure. The relative inefficiency of this response may be related to an expansion of a functionally and phenotypically distinct population of exhausted MBCs which we found to be associated with Pf exposure in Malian children and adults. B cells with a similar phenotype have also been identified in individuals infected with HIV and HCV. These cells express high levels of inhibitory receptors and a profile of lymphoid-homing receptors similar to what is expressed on exhausted CD8+ T cells during chronic viral infections. This observation provided insight into possible mechanisms by which the Ab response to malaria may be dysregulated. A major obstacle to understanding the origin, specificity and function of exhausted MBCs in the context of malaria is the small number of PBMCs that can be obtained through standard phlebotomy. To overcome this obstacle we are implementing an apheresis protocol in Mali which will permit a more comprehensive phenotypic and functional analysis of exhausted MBCs and other adaptive and innate immune response to malaria. In 2011 we also investigated the regulation of Pf-induced inflammation. PBMCs collected before the malaria season, seven days after the first malaria episode of the year, and after the following six-month dry season were analyzed. Genome-wide expression analysis revealed that recent symptomatic malaria primarily effected pattern recognition receptor signaling pathways, in particular, NALP3 and IL1-beta were significantly down-regulated. Total Pf-specific IL-10 responses in the supernatants were markedly upregulated after acute malaria but short-lived in the absence of ongoing Pf exposure. The majority of IL-10 was produced by foxp3- CD4+ T cells which also expanded after acute malaria and then returned to pre-infection levels after the following dry season. Despite a marked increase in the Pf-specific IL-10 response following malaria, the pro-inflammatory cytokine response remained constant before and after infection. Taken together, these data suggest a possible mechanism by which immunity to the inflammation-driven clinical manifestations of malaria are rapidly acquired during the malaria season and then lost in the absence of ongoing parasite exposure. In May 2011, to further elucidate the mechanisms of innate and acquired immunity to malaria, we initiated a cohort study in Mali of 695 individuals aged 3 months to 25 years. Sample collected from individuals prospectively classified as malaria immune and susceptible will be analyzed using a systems immunological approach to identify a signature of immunity to malaria. Signatures that correlate with protection from malaria may yield new hypotheses regarding the biological mechanisms through which malaria immunity is induced by natural Pf infection. We anticipate that the resulting datasets will be of considerable value in the urgent worldwide effort to develop a malaria vaccine.